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THE 1953 STANLEY L. MILLER EXPERIMENT: 
FIFTY YEARS OF PREBIOTIC ORGANIC CHEMISTRY 



ANTONIO LAZCANO'- • and JEFFREY L. BADA^ 
' Facidtadde Ciencias, UNAM Apdo. Postal 70-407, Cd. Universitaria, Mexico D.F., Mexico; 
^ Scripps Institution of Oceanography, University of California at San Diego, LaJolla, CA, U.S.A. 
(* author for correspondence, e-mail: alar@correoMnam.mx) 



(Received 19 March 2003) 



Abstract The field of prebiotic chemistry effectively began with a publication in Science 50 years 
ago by Stanley L. Miller on the spaik discharge synthesis of amino acids and odier compounds using 
a mixture of reduced gases that were thought to represent the components of the atmosphere on the 
primitive Earth. On the anniversary of this landmark publication, we provide here an accounting of 
the events leading to the publication of the paper. We also discuss the historical aspects that lead up 
to the landmark Miller experiment 

Keywords: prebiotic chemistry, reducing atmosf^ere, Stiecker sjrnthesis, electric discharges 



1. Introduction 

Fifty years ago. Science published in its 15 May 1953 issue the short, less than 
two-page, paper by Stanley L. Miller titled 'A production of amino acids under 
possible primitive Earth conditions' (Miller, 1953). In it, Stanley reported the stun- 
ning results he had achieved by the action of an electric discharge on a mixture of 
the reducing gases CH4, NH3, H2O, and H2 that simulated what was viewed at the 
time as a model atmosphere for the primitive Earth. The result of this experiment 
was a substantial yield of a mixture of amino acids, together with hydroxy acids, 
short aliphatic acids, and urea. One of the surprising results of this experiment 
was that the products were not a random mixture of organic compounds; rather, a 
relatively small number of compounds were produced in surprisingly high yields. 
Moreover, with a few exceptions, the compounds were of biochemical significance, 
thus providing support for the primitive soup heterotrophic theory projxjsed in the 
1920's by Oparin and Haldane. With this landmark experiment the modem era in 
the study of origin of life began. 



2. The Hectic Story Behind Publication 

Although the primitive soup theory had attracted considerable attention among bio- 
logists, it had gone largely unnoticed in other fields of science. In order to buttress 



M 



Origins of Life and Evolution of the Biosphere 33: 235-242,2003. 
© 2003 Kluwer Academic Publishers. Printed in the Netherlands. 



236 A. LAZCANO AND J. L. BADA 

his intuition, Oparin needed to demonstrate that organic compounds could form in 
the absence of living beings. Although he did not perform any actual experimental 
simulations of the primitive milieu, several important pieces of information suppor- 
ted his claim, including the universality of anaerobic fermentation and the existence 
of extraterrestrial organic compounds in meteorites, that the first organisms were 
more likely to have been heterotrophic (Oparin, 1938). 

One attempt to study the possibility of organic compound synthesis under prim- 
itive Earth conditions begun in 1950, when Melvin Calvin's group at the University 
of California, Berkeley, irradiated a gas mixture of COj, HnO, H2 and a solution 
of Fe-"*" with 40-meV helium ions in an attempt to simulate the radiation environ- 
ment in the terrestrial crust (Garrison et al, 1951). The results, however, were not 
encouraging: only small amounts of formic acid and formaldehyde were obtained, 
which is similar to results obtained in experiments done since the 1 920s by several 
other researchers (see Rabinowitch, 1945). 

Harold C. Urey, who had been involved with the study of the origin of the solar 
system and the chemical events associated with this process, would later consider 
the origin of life in the context of his proposal of a highly reducing terrestrial 
atmosphere. Urey first presented his ideas in a lecture at the University of Chicago 
in the fall of 1951, and the next year he published a paper detailing his model 
of the Earth's primitive atmosphere (Urey, 1952). In September 1952, almost year 
and a half after attending Urey's seminar, Stanley L. Miller, then a graduate student 
in Chemistry at the University of Chicago, approached Urey about the possibility 
of doing a prebiotic synthesis experiment using a reducing gas mixture (Miller, 
1974). After overcoming Urey's initial resistance, they designed three different 
spark discharge apparatus to be used in the experiment (Figure 1 ). The apparatus 
was meant to simulate the ocean-atmosphere system on the primitive Earth. Water 
vapor produced by heating would be like evaporation from the oceans, and as 
it mixed with methane, ammonia and hydrogen, it would mimic a water vapor- 
saturated primitive atmosphere. The apparatus shown in Figures la and b was 
the one most extensively used in the original experiments, and is the design most 
widely known today. The apparatus in Figure Ic led to a higher inner pressure, and 
an important aspect of this design is that it generated a hot water mist that could be 
considered similar to a water vapor-rich volcanic eruption. The apparatus shown in 
Figure Id used a so-called silent discharge instead of a spark, a concept that had 
been used previously in attempts to make organic compounds from CO2 in order 
to try to understand photosynthesis (Rabinowitch, 1945). 

Results were produced almost as soon as Stanley begun the experiments in 
the fall of 1952. Although by comparison with contemporary analytical tools the 
methods available to Stanley were crude, he was able to demonstrate that after 
only two days of sparking the gaseous mixture glycine could be detected. After 
repeating the experiment sparking the mixture for a whole week, the inside of the 
sparking flask was coated with an oily material and the water had a yellow-brown 
color. When paper chromatography was used to analyse the compounds that had 



RFTY YEARS OF PREBIOTIC ORGANIC CHEMISTRY 



237 



500-CC 
Flask 




-10 cm— »- 



Figure 1. The various qjparatus used in the Miller experiment (Miller, 1954; the photograph is 
courtesy of Stanley L. Miller). The design shown in (a) and (b) is the one that was used for the 
experiments published in Science on 15 May 1953. The apparatus shown in (c) was also tested and 
in general yielded similar results to those obtained with the one in (a). The apparatus in (d) used a 
silent discharge tiiat was generated by delivering ~97 watts of power to copper electrcxies placed in 
50% H2SO4 that filled die shaded area of the apparatus. 



238 



A. LAZCANO AND J. L. BADA 



PHENOL (0.3% NH3) 



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Figure 2. The two-dimensional paper chromatograin of the amino acids pnduced from the sparking 
experiment using lite apparatus shown in Figures la and b (courtesy of Staidey L. Miller). The spots 
corresponding the various amino acids were produced by spraying the chromatograin with ninhydrin. 
The amino acid labels are Stanley's original writing. We have adikd numbers to help identify the 
various spots. 1 = Aspartic acid; 2 = glycine; 3 = a-alanine; 4 = ^-alanine; and 5 = of-amino-n-butyric 
acid. Spots labeled A and B where not identified. 



formed, the glycine spot was much more than intense and spots corresponding to 
several other amino acids were also detected (Figure 2). 

Experiments with the apparatus in Figures 1 a and b as well as the one in Fig- 
ure Ic produced in general a similar distribution and quantities of amino acids 
and other organic compounds. In contrast, experiments with the apparatus in Fig- 
ure Id showed lower overall yields and a much more limited suite of amino acids: 
essentially only sarcosine and glycine were produced (Miller, 1955). 

After Miller showed the impressive results to Urey, they decided that it was 
time to get them published, preferably in a leading journal such as Science. Urey 
contacted the editors and asked for the paper to be published as soon as possible. 
He also declined Stanley's offer to coauthor the report because otherwise Stanley 
would receive little or no credit. In the meantime, Urey became so enticed by the 
outcome of the experiment that he began mentioning in his lectures the results 
achieved by Stanley. As shown by the articles published on 24 November 1952 
issues of both Time and Newsweek, the news attracted considerable attention not 
only from scientists but also from the media. Could it be that the origin of life could 
finally be understood? 

The manuscript was mailed to Science on 10 February 1953, and was received 
at the editorial office on 14 February (a detailed record of the submission and 
subsequent correspondence with Science is in the Urey papers in the Mandeville 
Special Collection at the University of California at San Diego library). On 27 
February 1953, Urey wrote Howard Meyeihoff, chairman of the Editorial Board, 



FIFTY YEARS OF PREBIOnC ORGANIC CHEMISTRY 239 

complaining about the lack of progress in publication of the manuscript. He stated 
'If Science does not wish to publish this promptly we will send it to the Journal 
of the American Chemical Society'. He closed the letter saying *I would appreciate 
an immediate reply so that we can make a decision in this matter'. 

In the meantime, on Sunday 8 March 1953, the Afeu- York Times published a 
rather cryptic short article titled 'Looking back two billion years', wherein the 
experiments of Wollman M. MacNevin and his associates at the Ohio State Uni- 
versity were described. It was reported that MacNevin and his team had performed 
a number of experiments simulating the primitive Earth, including a discharge ex- 
periment in which a spark was sent through methane producing 'resinous solids to 
complex for analysis'. MacNevin also reported the production of porphyrin from 
the heating of a mixture of CO2, H2O and NH3. The next day Stanley sent Urey 
a copy of the clipping together with a note in which he wrote 'I am not sure what 
should be done now, since their work [MacNevin and his group] is, in essence, my 
thesis. As of today, I have not received the proof from Science, and in the letter that 
was sent to you, Meyerhoff said that he had sent my note for review'. 

Infuriated by what he believed to be an unfair delay, Urey telegrammed Mey- 
erhoff on 10 March asking that Science return the paper. He then submitted the 
manuscript for Stanley to the Journal of the American Chemical Society on 13 
March. In the meantime, Meyerhoff, obviously frustrated with Urey, wrote directly 
to Stanley on 1 1 March telling him that he wanted to publish the manuscript and 
that he was 'unwilling to accept Dr. Urey's orders, unless it is your personal wish 
that the manuscript be returned to you and not used as a lead article in Science'. 
Stanley promptly accepted Meyerhoff's offer to publish the manuscript and tele- 
grammed the Editor of Journal of the American Chemical Society asking that the 
manuscript be returned, stating 'A mistake was made in sending this to you'. The 
paper appeared 2 months later in the 15 May issue of Science. 

Interestingly, while Stanley's manuscript was under review at Science, another 
paper by Kenneth Wilde and co-workers, on the attempted electric arc synthesis of 
organic compounds using CO2 and water was also under review. This manuscript 
was actually received on 15 December 1952, before Stanley's was submitted. In the 
Wilde et al., manuscript, it was reported that no interesting reduction products, such 
as formaldehyde, were synthesized using the C02/water mixture. This result nicely 
supported the surmise of Miller and Urey that reducing conditions were needed in 
order for effective organic syntheses to take place. The Wilde et al. (1953) paper 
was published in Science on 1 July 1 953, and made no mention of Stanley's paper 
although they did mention that their experiments had 'implications with respect to 
the origin of living matter on earth'. 



240 A. LAZCANOANDJ.L.BADA 

3. Earlier Laboratory Syntheses of Amino Acids 

Friedrich Wohler's report in 1828 on the synthesis of urea from silver cyanide 
and ammonium chloride represented the first synthesis of an organic compound 
from inorganic starting materials (Wohler, 1828). Although it was not immediately 
recognized as such, a new era in chemical research had begun: in 1850 Adolph 
Strecker achieved the laboratory synthesis of alanine from a mixture of acetal- 
dehyde, ammonia and hydrogen cyanide (Strecker, 1850). This was followed by 
the experiments of Alexandr M. Butlerov (1861a, b) showing that the treatment of 
formaldehyde with strong alkaline catalysts, such as sodium hydro.xide (NaOH), 
leads to the synthesis of sugars. 

The laboratory synthesis of biochemical compounds was soon extended to in- 
clude more complex experimental settings. By the end of the 19th century a large 
amount of research on organic synthesis had been performed, and had led to the 
abiotic formation of fatty acids and sugars using electric discharges with various 
gas mixtures (Rabinowitch, 1945). This work was continued into the 20th cen- 
tury by Klages (1903) and Ling and Nanji (1922), who reported the formation 
of glycine from formaldehyde and potassium cyanide, probably as a result of a 
Strecker synthesis and by Herrera (1942), who reported two uncharacterized amino 
acids using the same starting material. Moreover, Walther Lob, Oskar Baudish, 
and others worked on the synthesis of amino acids by exposing wet formamide 
(CHO-NHi) to a silent electrical discharge (Lob, 1913) and to UV light (Baudish, 
1913). 

Lob did indeed report the synthesis of glycine by exposing wet formamide to a 
silent discharge. He suggested that because of either the ultraviolet light or the elec- 
trical field generated by the silent discharge, formamide is first converted to oxamic 
acid, which in tum is reduced to glycine. He also claimed that glycine is produced 
when wet carbon monoxide and ammonia are subjected to the silent discharge; he 
proposed formamide as the intermediate in this synthesis. Lob theorized that gly- 
cine might also be produced from wet carbon dioxide and ammonia in a pathway 
wherein formamide was again the intermediate, but he did not demonstrate this 
directly. 

Although Lob apparently did produce glycine from formamide, this cannot be 
considered a prebiotic reaction because formamide would not have been present 
on the primitive Earth in any significant concentrations. It is also possible that the 
wet carbon monoxide and anmionia led to the formation of HCN, which would 
have produced glycine on polymerization and hydrolysis. From a careful reading 
of Lob's 1913 paper it is clear that his motivation for doing the exp>eriment was to 
try to understand the assimilation of carbon dioxide and nitrogen in plants. There 
is no indication that he had any interest in the question of how life began on Earth, 
or in the synthesis of organic compounds under possible prebiotic conditions. This 
is not surprising. Since it was generally assumed that that the first living beings had 
been autotrophic, plant-like organisms, the abiotic synthesis of organic compounds 



nFTY YEARS OF PREBIOTIC ORGANIC CHEMISTRY 24 1 

did not appear to be a necessary prerequisite for the emergence of life. With the 
exception of Herrera (1942), who tried to demonstrate the likelihood of an auto- 
trophic origin of life, these organic syntheses were not conceived as laboratory 
simulations of Danvin's warm little pond, but rather as attempts to understand the 
autotrophic mechanisms of nitrogen assimilation and COt fixation in green plants. 
Quite suiprisingly, in his extensive review Oparin (1938) did not mention neither 
Strecker's synthesis of alanine or Lob's work with electric discharges, which may 
have been forgotten by then. To the best of our knowledge, the work of Lob and 
other 19th century chemists was first discussed within the context of prebiotic 
chemistry by Stanley L. Miller in his 1954 Ph.D. Thesis and in an article following 
the publication of his 1953 Science paper (Miller, 1955). 

3.1. 1953: Annus mirabilis 

On 29 May 1953, Sir Edmund Hilary and his sherpa Tenzing Norgay, reached the 
summit of the Sagarmatha, as the Nepals call Mount Everest, the world's highest 
mountain. They thus achieved something that had seemed impossible. Other major 
peaks were also reached during the first several months of 1953, which, in retro- 
spect, have had a tremendous impact on our understanding of the origin and nature 
of life: Stalin died on 5 March, finally liberating genetics research in the USSR 
from the grasp of Trofim D. Lysenko; also in March, the first report on part of the 
amino acid sequence of a protein (insulin) was published by Sanger and Thompson 
(1953); on 25 April the double-helix model of DNA was published in Nature by 
Watson and Crick (1953); and of course Miller's publication on 15 May on the 
prebiotic synthesis of amino acids and organic compounds under plausible primor- 
dial conditions. These were major intellectual events whose importance cannot be 
underscored. 

The tremendous impact of the 1953 Miller experiment almost overnight trans- 
formed the study of the origin of life into a respectable field of inquiry, at a time in 
which not only the molecular nature of the genetic material was being elucidated 
by the models of Watson and Crick, but also the demonstration that amino acids 
are not randomly located in a protein was also shown. The times were ripe, as 
not only the effort of Wilde, Calvin and others to attempt oi^ganic synthesis under 
primitive conditions show, but also because by then, evolutionary biology was rap- 
idly becoming an established, properly recognized field of scientific enquire (Ruse, 
1999) that could accommodate quite easily the study of the origin of life, and by 
the development of space programs which would soon open new perspectives for 
those interested in the appearance of life in the Universe (Wolfe, 20O2). 

Although some of Lob's results as well as those of other 19th century organic 
chemists may have some bearing on our understanding of prebiotic syntheses, part 
of the significance of Miller's experiment lies not only in the production of amino 
acids and other compounds, but in their formation under what was viewed at the 
time as plausible primitive Earth conditions. Few, if any, scientific ideas are the 



242 A .LAZCANOANDJ.L.BADA 

product of spontaneous thoughts most theories, experiments and interpietations 
have been preceded by many others, and the same is true of Miller's experiment. 
Even if one disagrees with the assumptions underlying the simulation by Stanley L. 
Miller and Harold C. Urey of the primitive Earth, it deserves recognition not only 
because of its intrinsic merits, but also because it opened new avenues of empirical 
research into the origin of life. 

There are huge gaps in our understanding of the origin and early evolution 
of life, and it is not clear that a sufficient variety of organic compounds could 
have been synthesized on the primitive Earth, Other possible sources for organic 
molecules likely included meteorites, whose indigenous amino acids are due to 
reactions involving ammonia, hydrogen cyanide, and aldehydes/ketones just like 
in Miller's experiment. It is possible that the primitive atmosphere Avas not as re- 
ducing as Oparin, Urey and Miller believed. Nevertheless, for all our uncertainties 
regarding the emergence of life, a proper assessment of the significance of Stanley 
L. Miller 1953 experiments implies that it is part of the classics that have shaped 
contemporary science. 



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